5-Aza-CdR对胶质瘤细胞生长及LRRC4基因异常甲基化的影响  

Effects of 5-aza-2′-deoxycitydine on Proliferation of Glioma Cell Lines and Abnormal Methylation of LRRC4 Gene

作　　者：张祖萍[1,2] 武明花[1] 唐海林[1] 王蓉[1] 李丹 李小玲[1] 李桂源[1] 

机构地区：[1]中南大学肿瘤研究所,长沙410078 [2]中南大学湘雅医学院寄生虫学教研室,长沙410078

出　　处：《生物化学与生物物理进展》2009年第7期904-909,共6页Progress In Biochemistry and Biophysics

基　　金：国家重重点基础研究发展计划(973)资助项目(2006CB91052;2006CB910504);国家111计划资助项目(111-2-12);国家自然科学基金资助项目(30770825;30600224);湖南省自然科学基金资助项目(06JJ20080)~~

摘　　要：LRRC4是一个新发现的胶质瘤抑瘤基因,它在多种胶质瘤细胞系和胶质瘤组织表达缺失或下调,前期研究结果表明胶质瘤细胞和组织中LRRC4的编码区未发生突变、缺失或重排.为了获得LRRC4作为胶质瘤抑瘤基因的进一步证据,采用去甲基化制剂5-Aza-CdR处理LRRC4表达缺失的SF126和SF767胶质瘤细胞,MSP和RT-PCR检测表明,LRRC4的启动子在表达缺失的SF126和SF767细胞存在完全的甲基化,而5-Aza-CdR能逆转LRRC4启动子的甲基化状态,恢复LRRC4的表达.MTT法测定显示,5-Aza-CdR使SF126和SF767胶质瘤细胞增殖受到明显抑制,并呈时间和剂量的依赖性.同时流式细胞仪检测显示,5-Aza-CdR使SF126和SF767胶质瘤细胞周期阻滞于G0/G1期.因此,5-Aza-CdR能抑制胶质瘤细胞SF126和SF767增殖并干扰其细胞周期,LRRC4启动子异常甲基化是其在胶质瘤细胞中表达缺失的重要机制,5-Aza-CdR能逆转LRRC4基因的甲基化,恢复LRRC4的表达,为LRRC4作为胶质瘤去甲基化治疗的靶标提供了科学依据.LRR C4, leucine-rich repeat C4 protein, is a new member of leucine-rich repeat （LRR） superfamily. It is a novel tumor suppressor. LRRC4 inactivation is commonly found in glioma cell lines and primary glioma biopsies. Previous study did not find any genetic alteration of LRRC4 in primary glioma. In order to explore an alternative mechanism underlying inactivation of LRRC4 in glioma, glioma cell lines SF126 and SF767 were treated by methylase inhibitor 5-Aza-2＇-deoxycytidine （5-Aza-CdR）. Methylation-specific PCR was used to examine the methylation status changes of LRRC4 promoter in SF126 and SF767 cell lines. LRRC4 mRNA expression in SF126 and SF767 cell lines treated by 5-Aza-CdR was detected by RT-PCR. In addition, the effect on glioma cell lines cell growth and cell cycle of 5-Aza-CdR were assayed by MTT and FCM. The results indicated that LRRC4 promter was completely methylated in SF 126 and SF767 cells. However, LRRC4 promoter aberrant hypermethylation can be reversed and LRRC4 expression can be induced by 5-Aza-CdR. Moreover, 5-Aza-CdR displayed a growth inhitory effect on SF 126 and SF767 cells in a dose- and time-dependent manner after exposure to 5-Aza-CdR at different concentration for different time. FCM analysis showed that SF126 and SF767 cells cell cycles were blocked at G0/G1 phase after 5-Aza-CdR treatment for three days. Taken together, glioma cell lines SF126 and SF767 cell growth could be inhibited and cell cycles could be blocked by 5-Aza-CdR; promoter hypermethylation is the important mechanism of LRRC4 inacctivation in glioma cell lines; methylation can be reversed and LRRC4 expression can be induced by 5-Aza-CdR. All this suggest that LRRC4 may serve as a demethylation therapeutic target in glioma.

关 键 词：5-AZA-CDR 胶质瘤细胞 LRRC4基因 DNA甲基化 

分 类 号：Q75[生物学—分子生物学] R73[医药卫生—肿瘤]